Supported ionic liquids to purify microbial L-Asparaginase

abstract

L-Asparaginase (ASNase) is a versatile enzyme that converts L-asparagine into ammonia and aspartic acid. This enzyme has applications in the food industry and health sector. However, high purity ASNase is required, resulting in high production costs. Therefore, in this work, two supported ionic liquids (SILs), specifically silica modified with dimethylbutylpropylammonium chloride ([Si][N3114]Cl) or triethylpropylammonium chloride ([Si][N3222]Cl), were investigated as alternative adsorption materials to purify ASNase. Different conditions were evaluated to improve enzyme purity, including total protein content in the cell extract, contact time, and SIL/cell extract ratio (w/v). Under optimal conditions using [Si][N3114]Cl, a maximum ASNase purification of 6.1-fold is achieved in a single step, resulting from the preferential attachment of other proteins on [Si][N3114]Cl SIL. According to the results, hydrophobic interactions rule the selective adsorption of protein impurities from the cell extract by the SIL, thereby increasing the ASNase purification levels. This approach offers a significant advantage, not requiring the desorption and elution of the target enzyme, while envisioning the application of SILs in a flow-through elution approach. The protonation state of protein surface was calculated by computational analysis, revealing that positively charged amino acids such as arginine and lysine block the effective binding of the enzyme to the SILs. Overall, if properly designed, SILs are promising alternative supports for the downstream processing of ASNase from cell extracts.

keywords

PURIFICATION; EXPRESSION

subject category

Biotechnology & Applied Microbiology; Engineering

authors

Almeida, MR; Nunes, JCF; Pereira, MM; Bento, HBS; Pedrolli, DB; Santos-Ebinuma, VC; Neves, MC; Freire, MG; Tavares, APM

Groups

G5 - Biomimetic, Biological and Living Materials
G6 - Virtual Materials and Artificial Intelligence

Projects

CICECO - Aveiro Institute of Materials (UIDB/50011/2020)

CICECO - Aveiro Institute of Materials (UIDP/50011/2020)

Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)

acknowledgements

This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 (DOI10.54499/UIDB/50011/2020), UIDP/50011/2020 (DOI10.54499/UIDP/50011/2020) & LA/P/0006/2020 (DOI10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC) . Ana P. M. Tavares and Marcia C. Neves acknowledge FCT for the research contract CEECIND/2020/01867 (DOI: 10.54499/2020.01867.CEECIND/CP1589/CT0013) and CEECIND/00383/2017 (DOI 10.54499/CEECIND/00383/2017/CP1459/CT0031), respectively. Valeria C. Santos-Ebinuma acknowledges FAPESP for financial support through the project 2019/15493-9 and 2021/06686-6. Valeria C. Santos-Ebinuma and Danielle B. Pedrolli thank the National Council of Scientific and Technology Development (CNPq) for the fellowship grant no. 312463/2021-9 and 310023/2020-3, respectively. Matheus M. Pereira acknowledges the financial support of FCT, Portugal, within the projects DOI: 10.54499/UIDB/00102/2020 (Base funding) and DOI: 10.54499/UIDP/00102/2020 (Programmatic funding) . Joao C. F. Nunes acknowledges SPQ and FCT for the PhD fellowship SFRH/BD/150671/2020 (DOI 10.54499/SFRH/BD/150671/2020) .